In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1

PLOS ONE LAB PROTOCOL In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1 Marc Blanch-Asensio☯, Sourik Dey☯, Shrikrishnan Sankaran ID* Bioprogrammable Materials, INM—Leibniz Institute for New Materials Campus D2 2, Saarbrücken, Germany ☯ These authors contributed equally to this work. * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Blanch-Asensio M, Dey S, Sankaran S (2023) In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1. PLoS ONE 18(2): e0281625. https://doi.org/ 10.1371/journal.pone.0281625 Editor: Hari S. Misra, Bhabha Atomic Research Centre, INDIA Received: September 28, 2022 Abstract Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1. Accepted: January 27, 2023 Published: February 16, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0281625 Copyright: © 2023 Blanch-Asensio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All raw data including images and sequencing files related to results described in this paper have been added to the OSF data repository and can be accessed at this DOI DOI: 10.17605/OSF.IO/C6X3D. Introduction Lactobacilli are a group of Gram-positive bacteria of great importance to the food and healthcare industries with numerous strains identified as being beneficial for humans, and used as probiotics [1–3]. Furthermore, since they naturally colonize almost every site of the human body that hosts a healthy microbiome, e.g. the gastrointestinal tract [4,5], urogenital tracts [6], oral cavity [7] and nasal cavity [8], lactobacilli are an excellent foundational candidate for the development of live biotherapeutic products (LBPs) [9]. Beyond their natural health benefits, there is considerable interest in engineering them with heterologous genes for therapeutic applications like drug delivery [10,11] and mucosal vaccinations [12,13]. However, one of the crucial factors slowing down progress in lactobacilli engineering is difficulties in transforming them with exogenous DNA [14]. This is largely due to their thick and complex cell wall structures, which prevent successful bacterial transformation if the concentration of plasmid DNA is less than >1 μg [15]. To obtain such high plasmid DNA quantities, shuttle vectors are often used that can be amplified in intermediate hosts, predominantly E. coli [16]. To facilitate the construction of recombinant plasmids, several shuttle vectors have been identified, which can PLOS ONE | https://doi.org/10.1371/journal.pone.0281625 February 16, 2023 1 / 14 PLOS ONE Funding: This work was supported by a the Deutsche Forschungsgemeinschaft (DFG) Research grant [Project # 455063657 - https:// gepris.dfg.de/gepris/projekt/455063657] for M.B. A., the DFG Collaborative Research Centre, SFB 1027 [Project # 200049484 - https://gepris.dfg.de/ gepris/projekt/466932240] for S.S. and the LeibnizGemeinschaft’s Leibniz Science Campus on Living Therapeutic Materials [LifeMat - https://www. lsclifemat.de/] for S.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Gibson Assembly-based direct cloning in L. plantarum WCFS1 undergo stable replication in both the cloning host, E. coli and the relevant Lactobacilli strains [17–19]. Nevertheless, since E. coli is a Gram-negative bacterium that is phylogenetically distant from Lactobacillus genera, this strategy can lead to genetic sequence incompatibilities due to GC-content differences [20], DNA methylation [21], repetitive sequence insertions [22] and toxic protein buildup in the E. coli cloning host [23]. Alternatively, the Gram-positive lactic acid bacterium, Lactococcus lactis, can also be used as an intermediate host for recombinant plasmid construction [24]. However, the availability of functional replication origins in L. lactis is limited [25] and inclusion of additional broad-range replicons significantly increases the size of the plasmid. The excessive increase in the size of the plasmid might lead to segregational instability [26] and thereby limit the size of the heterologous genes that can be included in it. Hence, it is desirable to be able to directly transform circular plasmid dsDNA into the lactobacilli strains without relying on intermediate bacterial hosts like E. coli and L. lactis. To avoid the need for an intermediate host, Spath et al. developed a direct cloning approach based on the assembly of PCR-amplified DNA fragments by restriction digestion and ligation to obtain optimal quantities of circular dsDNA for transformation in Lactiplantibacillus plantarum CD033 [27]. They further show that the unmethylated plasmid DNA allowed for transformation in a strain (L. plantarum DSM20174) that could not be transformed using methylated DNA, possibly due to native restriction-modification systems. However, the method still requires the presence of restriction sites within the DNA sequences, which can limit the versatility of combining heterologous genes in the plasmid and needs to be accounted for when dealing with strains that may harbor unknown restriction-modification systems. In this work, we report a direct cloning method that leverages the Gibson assembly strategy and takes advan (...truncated)